How does ageing-related loss of basement membrane collagen regulate epidermal barrier homeostasis?

目的1建立含有Col 7或Col 17的shRNA敲低的永生化角质形成细胞的三维皮肤等效物。整合素受体，肌动蛋白，核细胞骨架和分化蛋白将使用免疫荧光检查。将挖掘来自具有Col 7和Col 17敲低（KD）的角质形成细胞的现有RNAseq数据以指导进一步分析。将使用染料排除、尼罗红染色和转氨酶活性测定来表征皮肤屏障。使用KD为Col 7/Col 17的原代（1 o）角质形成细胞重复实验。目标2对目标1 3D皮肤等效物施加机械/UV应力。学生将在可拉伸多孔膜（Gautrot实验室）上开发3D模型，以应用机械拉伸。另一种应激源是紫外线照射。同时，将使用Seahorse技术在单层培养物中测量糖酵解和氧化磷酸化，KD为Col 7/Col 17。将对氧化应激、增殖和DNA损伤标记物进行免疫染色。核形态和染色质重塑的变化将通过组蛋白标记的免疫染色和高分辨率共聚焦microscope.Aim 3使用表观基因组学方法来确定BM损失的转录反应的调节剂进行分析。使用选定转录调节因子的抗体对来自Aim 2的角质形成细胞进行染色质免疫沉淀（ChIP），然后进行下一代测序（NGS）。学生将这些数据与现有的RNAseq数据整合，以确定BM如何调节表皮稳态。结果可以比较在年轻和老年皮肤immunostaining.Aim 4识别和测试化合物，以减轻影响BM改变表皮homeostasAt联合利华，生物信息学和化学信息学工具将用于识别化合物，通过结合基于RNA的签名分析，路径驱动和目标/铅ID的方法。将在老化角质形成细胞模型中测试原理选项的证明，以检查屏障和基底膜是否可以改善。

Aim 1 Establish 3D skin equivalents incorporating immortalised keratinocytes with shRNA knockdown of Col7 or Col17. Integrin receptors, actin, nucleocytoskeleton and differentiation proteins will be examined using immunofluorescence. Existing RNAseq data from keratinocytes with knockdown (KD) of Col7 and Col17 will be mined to direct further analyses. The skin barrier will be characterized using dye exclusion, Nile Red staining and transglutaminase activity assays. Experiments will be replicated using primary (1o) keratinocytes with KD of Col7/Col17. Aim 2 Apply mechanical/UV stress to the Aim 1 3D skin equivalents. The student will develop the 3D models on a stretchable porous membrane (Gautrot lab) to apply mechanical stretch. An alternative stressor is UV irradiation. In parallel, glycolysis and oxidative phosphorylation will be measured using Seahorse technology in monolayer cultures with KD of Col7/Col17 . Immunostaining will be performed for oxidative stress, proliferation and DNA damage markers. Changes in nuclear morphology and chromatin remodelling will be analysed by immunostaining of histone marks and high-resolution confocal microscopy.Aim 3 Use epigenomic methods to identify regulators of the identified transcriptional response from BM loss. Keratinocytes from Aim 2 will be subjected to chromatin immunoprecipitation (ChIP) using antibodies for selected transcriptional regulators followed by Next Generation Sequencing (NGS). The student will integrate these data with the existing RNAseq data to identify how the BM is regulating epidermal homeostasis. Findings can be compared in young and aged skin by immunostaining.Aim 4 Identify and test compounds to mitigate effects of BM alterations on epidermal homeostasisAt Unilever, bioinformatic and chemi-informatic tools will be used to identify compounds via a combination of RNA-based signature analysis, pathway-driven and target/lead ID approaches. Proof of principle options will be tested in aged keratinocyte models to examine if the barrier and basement membrane can be improved.